CN102453767B - Method and kit for quickly and quantificationally detecting lactobacillus plantarum ST-III - Google Patents
Method and kit for quickly and quantificationally detecting lactobacillus plantarum ST-III Download PDFInfo
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Abstract
The invention discloses a method and a kit for quickly and quantificationally detecting lactobacillus plantarum ST-III. The method comprises the following steps of: 1) extracting total RNA (ribonucleic acid) of a sample to be detected; 2) carrying out inverse transcription on the total RNA extracted in the step 1) to obtain cDNA (complementary deoxyribonucleic acid); 3) carrying out fluorescent quantitative PCR (polymerase chain reaction) amplification detection by utilizing a primer pair of specifically amplified lactobacillus plantarum ST-III and taking the cDNA obtained in the step 2) as a template; and 4) comparing the circulation thresholds of the sample to be detected and a quantitative external standard product to quantify the actual number of copies in the sample to be detected. The method and the kit can quickly detect the quantity of lactobacillus plantarum ST-III in a detected sample and have fast speed, strong specificity and high sensitivity, and the limit of detection can be up to 2.85*10<3>CFU/mL.
Description
Technical field
The invention belongs to technical field of biological, particularly a kind of fast quantification detects method and the test kit thereof of plant lactobacillus ST-III.
Background technology
Probiotic bacterium is " after taking in some amount, can play the microorganism live body of beneficial effect to host health ".Along with subject development such as biotechnology and medical science, probiotic bacterium is confirmed more and more to the beneficial functional of HUMAN HEALTH, and probiotic composition also so day by day is subjected to pursuing of people, and production and sales volume are also in rising trend year by year.Studies show that to have only that the viable count of probiotic bacterium reaches 1 * 10 in the product in a large number
6Individual/ML can bring into play its due effect.Yet because the standard of probiotic composition is not still systematically studied and formulate in China; particularly do not carry out the strictness restriction to contained number of live bacteria of probiotics and at shelf-lives probiotic bacterium survival number on the market; cause probiotic bacterium and goods market chaotic, even appear at the phenomenon of mixing the spurious with the genuine that can not check this probiotic bacterium in the probiotic composition.So, to the supervision and management of its quality, especially the kind of its contained probiotic bacterium and the detection of quantity thereof have just been seemed particularly important.
To the qualitative and quantitative analysis of probiotic bacterium, still adopt the biochemical reactions method that pure culture separates counting with selective medium in the traditional method.These methods are subject to factor affecting such as culture condition, strain properties, and detection efficiency is limited, particularly be difficult to distinguish different bacterium of the same race, make the actual quantity that is difficult to detect the target probiotic bacterium in containing the complex system of various bacteria.Along with the progress of science and technology, be the base molecule biological method is successfully got involved in probiotic bacterium with advantages such as its higher accuracy and stability evaluation field with PCR.Particularly the appearance of real-time fluorescence quantitative PCR (Real-time PCR) has realized the leap of round pcr from qualitative to quantitative especially, and compare with conventional PCR, it have specificity stronger, effectively solve characteristics such as PCR pollution problem, level of automation height, become the important method of molecular biology and microbiology field detection by quantitative.Adopt the real-time fluorescence quantitative PCR technology to set up easy, probiotic bacterium quantitative detecting method in the probiotic bacteria milk product fast and accurately, can simplify trace routine, the raising detection efficiency of probiotic bacterium, can improve China probiotic bacterium the detection by quantitative ability, improve the quality monitoring of protective foods, and provide technical guarantee for the long term growth of probiotic bacterium industry.
Plant lactobacillus ST-III is a kind of from being the probiotic bacterium with multiple functions such as decreasing cholesterol, reducing blood-fat, adhesion intestinal epithelial cells that separation obtains from pickles.This bacterial strain is put into serial production listing as productive leavening agent in bright dairy industry company limited, and the market reflection is good.Give birth to function for the benefit of bringing into play ST-III better, just must guarantee that the product viable count reaches 1 * 10
6Individual/ML, this just needs to measure the actual quantity of ST-III in probiotic products.Therefore set up a kind of quick, easy, specifically in the testing product method of ST-III actual quantity for actual production with use significant.
Summary of the invention
Technical problem to be solved by this invention is at the present situation that does not still have at present plant lactobacillus ST-III bacterial strain Molecular Detection means, provide a kind of fast, method and the test kit thereof of high specificity, highly sensitive, detection by quantitative plant lactobacillus ST-III that cost is low.
One of technical scheme that the present invention takes is: a kind of fast quantification detects the method for plant lactobacillus ST-III, may further comprise the steps:
1) total RNA of extraction sample to be checked;
2) total RNA reverse transcription that step 1) is extracted becomes cDNA;
3) adopting the primer of specific amplification plant lactobacillus ST-III right, with step 2) gained cDNA is template, carries out fluorescent quantitative PCR and detects;
4) the actual copy number for the treatment of in the sample product by the cycle threshold of sample more to be checked and quantitative outer standard substance carries out quantitatively.
The described primer centering of step 3) of the present invention, a primer is identical with the sequence of the genetic marker molecule of plant lactobacillus ST-III bacterial strain, and primer length is 15-40bp, 20-30bp more preferably, that best is 23bp; The sequence complementation of the genetic marker molecule of another primer and plant lactobacillus ST-III bacterial strain, primer length are 15-40bp, 20-30bp more preferably, and that best is 27bp; The right amplified fragments of this primer is 200-867bp, preferred 242bp; Described plant lactobacillus ST-III genetic marker molecule is the gene of coded plant Bacterium lacticum ST-III KilA domain protein; Preferably the 1st of the KilA domain protein gene of coded plant Bacterium lacticum ST-III 200-867 continuous to the Nucleotide of sequence shown in the 867th, preferred 242 nucleotide fragments; Preferred, be the Nucleotide of sequence shown in the 360th to the 601st of KilA domain protein gene of coded plant Bacterium lacticum ST-III; Most preferred its nucleotide sequence is shown in the 360th to the 601st of SEQ ID NO.1.Most preferred, described primer centering, the sequence of a primer is shown in SEQ ID NO.2, and the sequence of another primer is shown in SEQ ID NO.3.
The described PCR reaction system of step 3) of the present invention is conventional, as long as can amplify the specificity product.The PCR reaction system preferably includes upstream primer, downstream primer, dNTP, PCR buffer, Mg
2+, the Taq archaeal dna polymerase, the cDNA template and
Green I.Wherein, the preferred final concentration of upstream primer or downstream primer is 0.1-1.0 μ M, more preferably 0.2 μ M; The final concentration of cDNA template is preferably 0.5-2ng/ μ L, preferred 1ng/ μ L.And other component concentrations such as routine generally are 0.2mmol/L dNTP, 1 * PCR buffer, 1.0mmol/L Mg
2+, 0.02U/ μ L Taq archaeal dna polymerase.The described PCR reaction parameter of step 3) is conventional, as long as can amplify the specificity product.Preferable is: 95 ℃ of 30S; 95 ℃ of 5S, 60 ℃ of 25S, 72 ℃ of 30S, 40 circulations; 4 ℃ of preservations.
Total cDNA after total RNA reverse transcription of the described quantitatively outer standard substance preferred plant Bacterium lacticum ST-III of step 4) of the present invention.Preferably, adopt quantitatively 10 times of gradient water diluents of outer standard substance, extent of dilution is from 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, 10
-8, carry out at quantitative fluorescent PCR, according to the cDNA molecular amounts in the quantitative sample to be checked of different Ct values of the quantitative criterion product of gradient dilution.
Two of the technical scheme that the present invention takes is: a kind of PCR kit for fluorescence quantitative that detects plant lactobacillus ST-III, comprise specific amplification plant lactobacillus ST-III genetic marker molecule primer right.This primer centering, a primer is identical with the sequence of the genetic marker molecule of plant lactobacillus ST-III bacterial strain, and primer length is 15-40bp, 20-30bp more preferably, that best is 23bp; The sequence complementation of the genetic marker molecule of another primer and plant lactobacillus ST-III bacterial strain, primer length are 15-40bp, 20-30bp more preferably, and that best is 27bp; The right amplified fragments of this primer is 200-867bp, preferred 242bp; Described plant lactobacillus ST-III genetic marker molecule is the gene of coded plant Bacterium lacticum ST-III KilA domain protein; Preferably the 1st of the KilA domain protein gene of coded plant Bacterium lacticum ST-III 200-867 continuous to the Nucleotide of sequence shown in the 867th, preferred 242 nucleotide fragments; Preferred, be the Nucleotide of sequence shown in the 360th to the 601st of KilA domain protein gene of coded plant Bacterium lacticum ST-III; Most preferred its nucleotide sequence is shown in the 360th to the 601st of SEQ IDNO.1.Most preferred, described primer centering, the sequence of a primer is shown in SEQ ID NO.2, and the sequence of another primer is shown in SEQ ID NO.3.
The PCR kit for fluorescence quantitative of detection plant lactobacillus ST-III of the present invention also comprises SYBR Green I fluorescent quantitation detection reagent.
SYBR Green I fluorescent quantitation detection reagent one preferred mode of the present invention contains the fluorescent quantitation reaction solution, comprising SYBR Green I fluorescence dye, PCR damping fluid, dNTPs solution, TaqDNA polysaccharase, aseptic double-distilled water, MgCl
2Components such as solution only need add primer and template and can carry out the PCR reaction during use.
Another preferred mode of SYBR Green I fluorescent quantitation detection reagent of the present invention contains fluorescent quantitation total reaction liquid, has wherein comprised SYBR Green I fluorescence dye, primer, PCR damping fluid, dNTPs solution, Taq archaeal dna polymerase, aseptic double-distilled water, MgCl
2All components such as solution only need add template and can carry out the PCR reaction during use, operate very quick and easy.
The PCR kit for fluorescence quantitative of detection plant lactobacillus ST-III of the present invention also comprises quantitatively outer standard substance.Total cDNA after total RNA reverse transcription that described quantitatively outer standard substance are plant lactobacillus ST-III.
The PCR kit for fluorescence quantitative of detection plant lactobacillus ST-III of the present invention comprises that also RNA extracts reagent and/or reverse transcription reagent.Described RNA extract reagent preferable contain Trizol, RNA enzyme inhibitors.Described reverse transcription reagent is preferable contains reversed transcriptive enzyme (AMV), AMV damping fluid.The AMV damping fluid contains dNTPs, random primer, RNA enzyme inhibitors, aseptic double-distilled water.
The raw material that the present invention is used or reagent except specifying, equal commercially available getting.
Among the present invention, plant lactobacillus ST-III is prior art, is CGMCC No.0847.
The invention provides a kind of method and test kit of rapid detection plant lactobacillus ST-III bacterial strain quantity, have following advantage:
1, fast, the high specificity of detection speed
Common biochemical identification for microorganism generally needs 2-3 days, and PCR identifies only needs 2-3 hour; And the different strains between the fubaritic kindred plant Bacterium lacticum of common biochemical identification, the PCR primer of the present invention design is to the specificity marker fragment design according to the KilA domain protein gene of coded plant Bacterium lacticum ST-III bacterial strain, this primer is to amplifying corresponding dna fragmentation in plant lactobacillus ST-III bacterial strain, and the gene of specific amplification plant lactobacillus ST-III only, and can not in other corresponding plants Bacterium lacticum, amplify respective segments, comprise the three kind of plant lactobacterium strains through genome sequencing that NCBI reported, illustrate with this gene fragment to be that the PCR reactive system of basic design has good specificity.
2, highly sensitive, cost is low
The growing amount of quantitative fluorescent PCR product increases with exponential manner, the template amplification initial to be measured of pieck stage can be arrived the microgram level, even only contain 3 target bacteria in the testing sample, PCR also can detect, and is therefore highly sensitive.Detectability of the present invention reaches 2.85x10
3CFU/mL.Common biochemical identification method agents useful for same kind is many, the step complexity, and PCR indentifying substance kind is few, simple to operate, cost is lower.In addition, compare with the method for different strains between bacterioid with distinguishing by whole genome sequence order-checking, expense greatly reduces, also shorten dramatically experimental period, can drop into practical application easily.
3, unique distinction of the present invention is, the design of special primer makes this method only increase to the characteristic fragment of ST-III, has got rid of the particularly interference of other plant Bacterium lacticum of other bacteriums; Can get rid of dead ST-III, the number of viable of a testing product based on the quantitative fluorescent PCR of RNA.
Description of drawings
Below in conjunction with description of drawings feature of the present invention and beneficial effect.
Fig. 1 is the quantitative fluorescent PCR typical curve of plant lactobacillus.
Embodiment
The inventor finds more suitable extension increasing sequence through research deeply and widely from the KilA domain protein gene of plant lactobacillus ST-III (lactobacillus plantarum ST-III).Wherein, the KilA domain protein is that a class DNA is in conjunction with albumen.This albumen is for being that dna replication dna, reorganization and reparation are most important.Select KilA domain protein gene order as primer, have good characteristic.
The three lactobacillus plantarum Lactobacillus plantarum WCFS1 (Genebank number:AL935263.1) that the present invention has reported according to NCBI, Lactobacillus plantarum 14917 (Genebank number:ACGZ00000000), the whole genome sequence of Lactobacillus plantarum JDM1 (Genebank number:CP001617.1), use the blast program to find out the exclusive nucleotide sequence of ST-III, design corresponding primer according to these nucleotide sequences.Through screening and the checking to these primers, obtained can be used in the Auele Specific Primer of plant identification Bacterium lacticum ST-III.Thus, the invention provides fluorescent quantificationally PCR detecting kit and the detection method of using this primer.The present invention to the detection of plant lactobacillus ST-III have fast, high specificity, highly sensitive, lower-cost characteristics.
The present invention finds the genetic marker molecule of a kind of plant lactobacillus (Lactobacillus plantarum) ST-III bacterial strain, and it is the gene of coded plant Bacterium lacticum ST-III KilA domain protein.KilA domain protein (KilA domain protein) is that a class DNA is in conjunction with albumen.The nucleotide sequence of the gene of coded plant Bacterium lacticum ST-III KilA domain protein preferably GenBank AccessionNo. is YP_003924036.1.Preferably, described genetic marker molecule is 200-867 continuous in the Nucleotide of sequence shown in the 1st to the 867th of KilA domain protein gene of coded plant Bacterium lacticum ST-III, preferred 242 nucleotide fragments.Preferred, described genetic marker molecule is the Nucleotide of sequence shown in the 360th to the 601st of KilA domain protein gene of coded plant Bacterium lacticum ST-III.Most preferred its nucleotide sequence of described genetic marker molecule is shown in the 360th to the 601st of SEQ ID NO.1.
It is right that the present invention has also invented the primer of a kind of detection plant lactobacillus (Lactobacillus plantarum) ST-III bacterial strain.This primer centering, a primer is identical with the sequence of the genetic marker molecule of plant lactobacillus ST-III bacterial strain, and primer length is 15-40bp, 20-30bp more preferably, that best is 23bp; The sequence complementation of the genetic marker molecule of another primer and plant lactobacillus ST-III bacterial strain, primer length are 15-40bp, 20-30bp more preferably, and that best is 27bp.The right amplified fragments of this primer is 200-867bp, preferred 242bp.Preferably, described primer centering, the sequence of a primer is shown in SEQID NO.2, and the sequence of another primer is shown in SEQ ID NO.3.
The primer of specific amplification plant lactobacillus ST-III bacterial strain of the present invention, sequence (SEQ ID NO.1) according to genetic marker of the present invention designs, and by conventional DNA synthetic method acquisition, for example can be synthetic with business-like automatic dna synthesizer.
Quantitative detecting method
Primer of the present invention is combined with the fluorescence dye technology, has just obtained fluorescent quantitative PCR detection method and the test kit thereof of plant lactobacillus ST-III of the present invention.This method has not only overcome the error in the conventional PCT method, and it is few to analyze required sample size, and detection limit is low, and is highly sensitive.
In order to realize the function quantitative to plant lactobacillus ST-III, use quantitative fluorescent PCR.Preferred chimeric fluorescence dye method, the employing fluorescence dye SYBR Green I of adopting.SYBR Green I is the most frequently used dna binding dye of quantitative fluorescent PCR, with the double-stranded DNA non-specific binding.Under unbound state, SYBR Green I sends faint fluorescence, but in case is combined 1000 times of its fluorescence increases with double-stranded DNA.So whole fluorescent signals that reaction is sent are than row with the double-stranded DNA amount of appearance, and can increase with the increase of amplified production.In the PCR reaction process, be combined with the double-stranded DNA that amplification produces as SYBR Green I, by measuring fluorescence intensity, can determine the content of double-stranded DNA.SYBR Green I particularly can be used for all PCR reaction systems, need not fluorescent probe, makes detection method become easy, has also reduced the cost that detects simultaneously.And after finishing, PCR can directly carry out the melt curve analysis response analysis.By the analysis of melt curve analysis, can judge whether to exist variation or nonspecific amplification.The maximum absorption wavelength of SYBR Green I is about 497nm, and the emission wavelength maximum is about 520nm.
The invention provides the method that a kind of fast quantification detects plant lactobacillus ST-III, may further comprise the steps:
1) total RNA of extraction sample to be checked;
2) total RNA reverse transcription that step 1) is extracted becomes cDNA;
3) adopting the primer of specific amplification plant lactobacillus ST-III right, with step 2) gained cDNA is template, carries out fluorescent quantitative PCR and detects;
4) the actual copy number for the treatment of in the sample product by the cycle threshold of sample more to be checked and quantitative outer standard substance carries out quantitatively.
Testing sample of the present invention is any material that may contain plant lactobacillus ST-III bacterial strain, and is preferable such as probiotic bacterium class cultured milk prod, comprises sour milk, also comprises fermenting bean milk etc.
Among the present invention, the method that step 1) is extracted total RNA of sample to be checked is routine techniques, as the Trizol extraction method, is well known to those skilled in the art.
Among the present invention, step 2) total RNA reverse transcription being become the method for cDNA also is routine techniques, generally react with ThermoScript II, generally on the PCR instrument under the suitable temperature of ThermoScript II reaction for some time get final product, this also is the method for being familiar with for those skilled in the art.
Among the present invention, step 3) adopts Auele Specific Primer of the present invention to carry out fluorescent quantitative PCR, and preferable carries out in the real-time fluorescence PCR instrument.Used primer for specific amplification plant lactobacillus ST-III genetic marker molecule that the present invention provided especially primer right.This primer centering, a primer is identical with the sequence of the genetic marker molecule of plant lactobacillus ST-III bacterial strain, and primer length is 15-40bp, 20-30bp more preferably, that best is 23bp; The sequence complementation of the genetic marker molecule of another primer and plant lactobacillus ST-III bacterial strain, primer length are 15-40bp, 20-30bp more preferably, and that best is 27bp.The right amplified fragments of this primer is 200-867bp, preferred 242bp.Described plant lactobacillus ST-III genetic marker molecule is the gene of coded plant Bacterium lacticum ST-III KilA domain protein; Preferably the 1st of the KilA domain protein gene of coded plant Bacterium lacticum ST-III 200-867 continuous to the Nucleotide of sequence shown in the 867th, preferred 242 nucleotide fragments; Preferred, be the Nucleotide of sequence shown in the 360th to the 601st of KilA domain protein gene of coded plant Bacterium lacticum ST-III; Most preferred its nucleotide sequence is shown in the 360th to the 601st of SEQ ID NO.1.Most preferred, described primer centering, the sequence of a primer is shown in SEQ IDNO.2, and the sequence of another primer is shown in SEQ ID NO.3.Their expanding fragment length is 242bp.
Wherein, described PCR reaction system is conventional, as long as can amplify the specificity product.The PCR reaction system preferably includes upstream primer, downstream primer, dNTP, PCR buffer, Mg
2+, the TaqDNA polysaccharase, the cDNA template and
Green I.Wherein, the final concentration of upstream primer or downstream primer is 0.1-1.0 μ M, preferred 0.2 μ M; The final concentration of cDNA template is 0.5-2ng/ μ L, preferred 1ng/ μ L.And other component concentrations such as routine generally are 0.2mmol/L dNTP, 1 * PCRbuffer, 1.0mmol/L Mg
2+, 0.02U/ μ L Taq archaeal dna polymerase.The described PCR reaction parameter of step 3) is conventional, as long as can amplify the specificity product.Preferable is: 95 ℃ of 30S; 95 ℃ of 5S, 60 ℃ of 25S, 72 ℃ of 30S, 40 circulations; 4 ℃ of preservations.
The actual copy number that step 4) is treated in the sample product by the cycle threshold of sample more to be checked and quantitative outer standard substance carries out quantitatively.Total cDNA after total RNA reverse transcription that described quantitatively outer standard substance are plant lactobacillus ST-III.Adopt quantitatively 10 times of gradient water diluents of outer standard substance, extent of dilution is from 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, 10
-8, carry out at quantitative fluorescent PCR, because cDNA molecule difference in each extent of dilution, the Ct value that obtains is difference just, according to cDNA molecular amounts such as the copy number in the quantitative sample to be checked of different Ct values of the quantitative criterion product of gradient dilution.
PCR kit for fluorescence quantitative
This test kit has adopted the chimeric fluorescent method of SYBR Green I to carry out the special agent of Real Time PCR, can carry out detection by quantitative to goal gene fast.In a preference, the invention provides the PCR kit for fluorescence quantitative of rapid detection plant lactobacillus ST-III, this test kit comprises that the primer of specific amplification plant lactobacillus ST-III genetic marker molecule is right.This primer centering, a primer is identical with the sequence of the genetic marker molecule of plant lactobacillus ST-III bacterial strain, and primer length is 15-40bp, 20-30bp more preferably, that best is 23bp; The sequence complementation of the genetic marker molecule of another primer and plant lactobacillus ST-III bacterial strain, primer length are 15-40bp, 20-30bp more preferably, and that best is 27bp.The right amplified fragments of this primer is 200-867bp, preferred 242bp.Described plant lactobacillus ST-III genetic marker molecule is the gene of coded plant Bacterium lacticum ST-III KilA domain protein; Preferably the 1st of the KilA domain protein gene of coded plant Bacterium lacticum ST-III 200-867 continuous to the Nucleotide of sequence shown in the 867th, preferred 242 nucleotide fragments; Preferred, be the Nucleotide of sequence shown in the 360th to the 601st of KilA domain protein gene of coded plant Bacterium lacticum ST-III; Most preferred its nucleotide sequence is shown in the 360th to the 601st of SEQ ID NO.1.Most preferred, described primer centering, the sequence of a primer is shown in SEQ ID NO.2, and the sequence of another primer is shown in SEQ ID NO.3.
In another preference, the PCR kit for fluorescence quantitative that fast quantification of the present invention detects plant lactobacillus ST-III comprises SYBR Green I fluorescent quantitation detection reagent.One preferred mode of described SYBR Green I fluorescent quantitation detection reagent is to contain the fluorescent quantitation reaction solution, comprising SYBR Green I fluorescence dye, PCR damping fluid, dNTPs solution, Taq archaeal dna polymerase, aseptic double-distilled water, MgCl
2Components such as solution only need add primer and template and can carry out the PCR reaction during use.Another preferred mode of described SYBRGreen I fluorescent quantitation detection reagent is to contain fluorescent quantitation total reaction liquid, has wherein comprised SYBR Green I fluorescence dye, primer, PCR damping fluid, dNTPs solution, Taq archaeal dna polymerase, aseptic double-distilled water, MgCl
2All components such as solution only need add template and can carry out the PCR reaction during use, operate very quick and easy.
Fluorescence can be detected the accumulation volume relation in direct ratio of the increase of fluorescence volume and PCR product by the fluorescent signal acquisition system in the quantitative PCR instrument.To plant lactobacillus ST-III quantitatively can comparing draws by the cycle threshold (Ct, Threshold Cycle) with the quantitative criterion product.The Ct value is in the PCR process, and the accumulation of fluorescence volume surpasses the cycle number of substrate fluorescence volume.Ct value and starting template are counted proportion relation, and the Ct value is more little, and the starting template number is more many; On the contrary, the Ct value is more big, and the starting template number is more few.Utilize the Ct value of gradient dilution standard substance to make typical curve, can accurately measure the initial copy number of this sample again according to the Ct value of testing sample.
Fast quantification provided by the invention detect the PCR kit for fluorescence quantitative of plant lactobacillus ST-III preferable also comprise quantitatively outer standard substance.Total RNA that described quantitatively outer standard substance are plant lactobacillus ST-III calculates its actual copy number through the absorbancy of measuring 260nm, and the total cDNA after the back reverse transcription adopts 10 times of gradient water diluents in the time of quantitatively, and extent of dilution is from 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, 10
-8In quantitative fluorescent PCR, because cDNA molecule difference in each extent of dilution, the Ct value that obtains is just different, according to the cDNA molecule in quantitative criterion product (the quantitative outer standard substance of gradient dilution) the quantitative sample of Ct value.
The composition that the two-step approach RT-PCR SYBR Green I fluorescence quantitative detection kit that also comprises routine that this test kit is preferable comprises extracts reagent, reverse transcription reagent as comprising RNA.Wherein preferable RNA extraction reagent contains Trizol, RNA enzyme inhibitors.Reverse transcription reagent contains reversed transcriptive enzyme (AMV), AMV damping fluid.The AMV damping fluid contains dNTPs, random primer, RNA enzyme inhibitors, aseptic double-distilled water.
The PCR kit for fluorescence quantitative of detection plant lactobacillus ST-III of the present invention is by to each component, as primer concentration, Mg
2+The optimization of concentration, annealing temperature etc., experiment repeatedly, test kit can satisfy the actual demand of quick special detection plant lactobacillus ST-III fully.
Further specify the present invention with embodiment below, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
The quantitative detecting method of plant lactobacillus in embodiment 1 probiotic bacteria milk product
A. the preparation of the total RNA of sample
Adopt the Trizol method to extract: to contain plant lactobacillus ST-III leben (bright smooth excellent plant lactobacillus drink) as implementing sample with what buy on the market, be respectively apart from the date manufactured be 19 days, three samples of 15 days and 13 days, place the mortar that fills liquid nitrogen to grind, add 1mLTrizol reagent then rapidly, press Trizol test kit specification sheets (
Plus RNA Purification System, U.S. invitrogen company, article No. 12183-555) extracts sample RNA, handle twice with DNaseI then, the DNA that guarantees to eliminate fully among the RNA pollutes, obtain the purified RNA sample, survey concentration, the RNA integrity detects by 1% agarose gel electrophoresis.
B. post transcription cloning
Carry out the RNA post transcription cloning according to reverse transcription test kit (M-MLV RTase cDNA Synthesis Kit, the precious biotechnology in Dalian company limited, article No. D6130) specification sheets.
C. real-time fluorescence quantitative PCR
Quantitative fluorescent PCR according to
PrimeScript
TMRT-PCR Kit (the precious biotechnology in Dalian company limited, article No. DRR063A) specification sheets carries out.Reaction system 25.0 μ L:
Ex Taq
TM(2 *) 12.5 μ L, each 0.5 μ L of 10 μ mol/L upstream and downstream primers (being respectively SEQ ID NO.2 and SEQID NO.3), cDNA template 2.0 μ L (reaction density 1ng/ μ L), ddH
2O 9.5 μ L.
Quantitative fluorescent PCR reaction parameter: 95 ℃ of 30S; 95 ℃ of 5S, 60 ℃ of 25S, 72 ℃ of 30S, 40 circulations; 4 ℃ of preservations.
D. the quantitatively preparation of outer standard substance and the drafting of typical curve
Extract total RNA of plant lactobacillus ST-III, after ultraviolet spectrophotometer is measured absorbance (260nm), calculate copy number according to the genome size of ST-III.Reverse transcription obtains cDNA, does 10 times of serial dilutions, carries out as above quantitative fluorescent PCR reaction with this as masterplate.Logarithm with the positive template of different copy numbers is X-coordinate, is the typical curve that ordinate zou obtains plant lactobacillus ST-III with the initial cycle number (Ct) that arrives fluorescence threshold in the PCR reaction process, sees Fig. 1.
The minimum concentration of dilution is 2.85x10
3CFU/mL is lower than this concentration, and fluorescent signal is mixed and disorderly, thereby the detection of this method is limited to 2.85x10
3CFU/mL.
E. result and judgement
CDNA with testing sample is template, with the bacterial strain Auele Specific Primer of plant lactobacillus ST-III, carries out the fluorescent quantitative PCR of plant lactobacillus characteristic fragment with identical system, the results are shown in Table 1.
According to the Ct value of formula: Y=-3.369lgX+39.84 and testing sample, can calculate the quantity of plant lactobacillus ST-III in the sample, see Table 1.
The quantitative result of ST-III content in table 1. product
Should be understood that after having read foregoing of the present invention, those skilled in the art can make various changes or modifications the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (7)
1. the method for a fast quantification detection plant lactobacillus ST-III is characterized in that, may further comprise the steps:
1) total RNA of extraction sample to be checked;
2) total RNA reverse transcription that step 1) is extracted becomes cDNA;
3) adopt the primer of specific amplification plant lactobacillus ST-III right, with step 2) gained cDNA is template, carries out fluorescent quantitative PCR and detects described primer centering, article one, the sequence of primer is shown in SEQ ID NO.2, and the sequence of another primer is shown in SEQ ID NO.3;
4) the actual copy number for the treatment of in the sample product by the cycle threshold of sample more to be checked and quantitative outer standard substance carries out quantitatively.
2. the method for claim 1, it is characterized in that, total cDNA after total RNA reverse transcription that the described quantitatively outer standard substance of step 4) are plant lactobacillus ST-III, adopt quantitatively that 10 times of gradient water diluents of outer standard substance carry out at quantitative fluorescent PCR, according to the cDNA molecular amounts in the quantitative sample to be checked of different Ct values of the quantitative criterion product of gradient dilution.
3. PCR kit for fluorescence quantitative that detects plant lactobacillus ST-III, it is characterized in that, comprise specific amplification plant lactobacillus ST-III genetic marker molecule primer right, described primer centering, article one, the sequence of primer is shown in SEQ ID NO.2, and the sequence of another primer is shown in SEQ ID NO.3; Described plant lactobacillus ST-III genetic marker molecule is the gene of coded plant Bacterium lacticum ST-III KilA domain protein.
4. PCR kit for fluorescence quantitative as claimed in claim 3, it is characterized in that, also comprise the fluorescent quantitation reaction solution in the described PCR kit for fluorescence quantitative, comprising SYBR Green I fluorescence dye, PCR damping fluid, dNTPs solution, Taq archaeal dna polymerase, aseptic double-distilled water and MgCl
2Solution.
5. PCR kit for fluorescence quantitative as claimed in claim 3, it is characterized in that, comprise fluorescent quantitation total reaction liquid in the described PCR kit for fluorescence quantitative, wherein comprised SYBR Green I fluorescence dye, primer, PCR damping fluid, dNTPs solution, Taq archaeal dna polymerase, aseptic double-distilled water and MgCl
2Solution.
6. PCR kit for fluorescence quantitative as claimed in claim 3 is characterized in that, described PCR kit for fluorescence quantitative also comprises quantitatively outer standard substance.
7. PCR kit for fluorescence quantitative as claimed in claim 3 is characterized in that, described PCR kit for fluorescence quantitative comprises that also RNA extracts reagent and/or reverse transcription reagent.
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| CN102994644B (en) * | 2012-12-31 | 2014-01-22 | 光明乳业股份有限公司 | Lactobacillus plantarum quantitative detection method and detection kit and application thereof |
| CN103525914B (en) * | 2013-09-23 | 2015-06-17 | 光明乳业股份有限公司 | Method, primer and kit for counting number of live bacteria of lactobacillus plantarum |
| KR101791570B1 (en) | 2015-10-28 | 2017-11-02 | 대한민국 | Lactobacillus plantarum specific primer and method for detecting Lactobacillus plantarum using thereof |
| CN106755470A (en) * | 2017-01-16 | 2017-05-31 | 吉林省浦生泰生物技术有限责任公司 | A kind of method of probiotics species and content in utilization Q PCR detections mixing probiotics |
| CN110982758A (en) * | 2019-12-30 | 2020-04-10 | 光明乳业股份有限公司 | Selenium-enriched lactobacillus preparation and preparation method thereof |
| CN110951652A (en) * | 2019-12-30 | 2020-04-03 | 光明乳业股份有限公司 | Selenium-enriched lactobacillus preparation and preparation method thereof |
| CN111088185A (en) * | 2019-12-30 | 2020-05-01 | 光明乳业股份有限公司 | Selenium-enriched lactobacillus preparation and preparation method thereof |
| CN110951651A (en) * | 2019-12-30 | 2020-04-03 | 光明乳业股份有限公司 | Selenium-enriched lactobacillus preparation and preparation method thereof |
| CN113755562B (en) * | 2021-10-20 | 2022-07-12 | 赛宁(苏州)生物科技有限公司 | Detection method of PCR inhibitor residue of biological consumable |
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| CN101712989B (en) * | 2009-09-21 | 2011-08-31 | 内蒙古农业大学 | Method for quickly, qualitatively and quantitatively measuring Lactobacillus casei in probiotic dairy products |
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